The number of Na-transporters in the cell membrane is regulated by dynamic insertion, retrieval and, recycling of transporters. Collectively these processes are known as trafficking. In some forms of human and experimental hypertension salt retention is caused by abnormally high levels of Na-transporters in the apical membrane of some nephron segments. The thick ascending limb (THAL) absorbs NaCI and contributes to salt and water homeostasis, thereby influencing blood pressure. NaCI absorption by the THAL is primarily regulated by NKCC2, an apical Na/K/2CI cotransporter that mediates Na and Cl entry into the cell. To date, it is not clear whether trafficking of NKCC2 regulates NaCI absorption. It is also not clear whether vasoactive hormones that stimulate and autacoids that inhibit NaCI absorption by the THAL regulate NKCC2 levels in the apical membrane by affecting its trafficking. We hypothesize that NaCI absorption by the THAL is regulated in part by trafficking of NKCC2 into the apical membrane, in a process stimulated by cAMP and inhibited by nitric oxide. Enhanced insertion of NKCC2 into the apical membrane contributes to salt retention in models of salt-sensitive hypertension. In Aim I we will examine whether NKCC2 levels in the apical membrane and NaCI absorption are regulated by insertion and retrieval of NKCC2. In Aim II we will study whether hormones that stimulate NKCC2-dependent NaCI entry into THALs via cAMP increase NKCC2 levels in the apical membrane. In Aim III we will study whether nitric oxide inhibits NKCC2-dependent NaCI entry by decreasing NKCC2 levels in the apical membrane. Finally we will examine whether basal and hormone-stimulated NKCC2 levels in the apical membrane are enhanced in THALs from salt-sensitive animals. We will use state-of-the-art techniques to measure NKCC2 trafficking and activity in isolated rat THALs. Data from this proposal will increase our understanding of how salt absorption by the THAL is regulated in normal and salt-sensitive animals, and will identify new targets for the development of diuretics.